Novel monooxygenase biocatalysts from the environment and the laboratory

Completed

Not Energy Related
Renewable Energy Sources(Bio-Energy, Production of transport biofuels (incl. Production from wastes))
Renewable Energy Sources(Bio-Energy, Production of other biomass-derived fuels (incl. Production from wastes))

Basic and strategic applied research

BIOLOGICAL AND AGRICULTURAL SCIENCES (Biological Sciences)

Not Cross-cutting

Dr TJ Smith
Faculty of Health and Wellbeing
Sheffield Hallam University

Research Grant

BBSRC

01 November 2008

31 October 2011

36 months

£293,906

Manufacturing

East Midlands

Dr TJ Smith, Faculty of Health and Wellbeing, Sheffield Hallam University

This grant is linked to BB/F012713/1.

This is an ambitious grant proposal to study the structure and function of the soluble, methane monooxygenase enzyme (sMMO) from methanotrophs. sMMO is an extremely versatile biocatalyst. Its biological function is to catalyse the first step in methane oxidation, methane to methanol. However, this enzyme is remarkable in that it also co-oxidises over 150 alkanes, alkenes, aromatic compounds and their substituted derivatives, making it an extremely versatile biocatalyst with huge industrial potential for green chemistry reactions in biotransformation and bioremediation. This catalytic versatility is of great interest to us and we seek to define the structure of the active site and to elucidate how substrates enter the active site, define the interactions of subunits that enable this wide substrate range and investigate how hydrogen tunnelling occurs and how intermediates in the oxidation of methane are formed. This will be achieved by the construction and analysis of a wide range of sMMO mutants using a homologous expression system that we developed which enables us to generate large quantities of highly active sMMO proteins. We will couple this with a unique approach involving DNA-Stable Isotope probing, pioneered in our lab for the analysis of active methanotroph populations in the environment. This gives immediate access to MMO enzymes and related di-iron centre monooxygenases present in the uncultivated majority of methanotrophs and related organisms in the environment. This form of gene mining gives us access to unique MMO gene sequences and their homologs which can then be amplified by PCR and cloned into our sMMO expression vectors, thereby generating new and novel biocatalysts based on sMMO which may have huge potential in biotransformation and bioremediation reactions. Analysis of these recombinant sMMOs, which may have considerable industrial potential, will also reveal important features which define the catalytic utility of this fascinating enzyme.

22/11/13